Isolated nucleic acid molecules encoding the Dw3 P-glycoprotein of sorghum and methods of modifying growth in transgenic plants therewith

ABSTRACT

The invention relates to the genetic manipulation of organisms, particularly to the expression of P-glycoprotein genes in transformed plants and other organisms. Nucleotide sequences for the P-glycoprotein genes, particularly the Dw3 gene of sorghum, and methods for their use are provided. The sequences find use in modifying the growth of organisms, particularly plants. Additionally, the invention provides methods for producing stable dwarf crop plants, particularly stable dwarf sorghum plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/711,619, filed Nov. 13, 2000, and claims the benefit of U.S.Provisional Application No. 60/165,176, filed Nov. 12, 1999; both ofwhich are hereby incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the genetic manipulation oforganisms, particularly plants, with genes that control growth anddevelopment. The invention further relates to genes that control growth,including homologues and mutant forms, the proteins encoded therefromand plants transformed with these genes.

BACKGROUND OF THE INVENTION

[0003] Dwarf plants have had a major impact on agriculture. Dwarfvarieties of wheat are widely used in North America due to both reducedpotential for lodging and high yields. Dwarf fruit trees are alsoextensively used and allow farmers to produce more fruit per acrethereby increasing economic yield potential. There are other benefitsthat may be realized from the use of dwarf crop plants and dwarf fruittrees including reductions in the amounts of pesticides and fertilizersrequired, higher planting densities and reduced labor costs.

[0004] In view of the current trends of both increasing human populationand the decreasing land area suitable for agriculture, increasingagricultural productivity is, and will continue to be, a challenge ofparamount importance. Dwarf crop plants and fruit trees have been andwill continue to be important components of our agricultural productionsystem. Increased usage of dwarf crop plants and dwarf fruit trees mayhelp to meet the agricultural production demands of the future. However,commercially acceptable dwarf varieties are not available for all crops.

[0005] In addition to the use of dwarf plants to control plant height,synthetic chemicals are routinely applied to certain economicallyimportant plant species to reduce growth. Plant growth regulators knownas growth retardants are used to reduce stem elongation in a variety ofcrops including cotton, grape vines, fruit trees, peanuts, wheat andornamentals such as azaleas, chrysanthemums, hydrangeas, poinsettias andmany bedding plants. All of the commonly used growth retardants areinhibitors of gibberellin biosynthesis and limit stem or shoot growth byreducing elongation. In the United States, the most widely used growthretardant is mepiquat chloride, which is registered for use on cotton.Benefits attributed to the use of mepiquat chloride on cotton includeincreased yield, improved defoliation, improved stress tolerance, moreuniform crop maturity and the ability to harvest earlier. Previously,the growth retardant daminozide was registered for use in the UnitedStates on apples, grapes and peanuts under the trademarks ALAR and KYLARbut was removed from use on food crops due to human health concerns.Despite the demands of agricultural producers for a product to replacediaminozide, there are no growth retardants registered for use ongrapes, fruit trees and peanuts in the United States. Daminozide,however, is still widely used on certain non-food, plant species.

[0006] Uncovering the molecular mechanisms that control plant growthprocesses such as cell division and cell elongation will likely aid inthe development of new plant varieties with reduced stature and newmethods for reducing plant growth. Such new plant varieties and methodsmay provide both farmers and horticulturists with environmentally benignalternatives to the use of synthetic growth-retarding chemicals.

[0007] Elongation of plant cells and organs is one of the most criticalparameters of plant growth and development. Regulation of this trait inplants, however, is a fairly complicated process, as both external andinternal factors influence it. The most important external stimulus islight, with its normally repressible or negative effect on cellelongation (Quail, P. H. (1995) Science 268:675-680; Kende et al. (1997)Plant Cell 9:1197-1210). The internal control of cell elongation ismediated by a number of chemicals, normally referred to as plant growthregulators or hormones (Kende et al. (1997) Plant Cell 9:1197-1210).Among the classical plant hormones, auxins and gibberellins (GAs) bothpromote cell elongation whereas cytokinins and abscisic acid each havebeen shown to have a negative effect on cell elongation (Kende et al.(1997) Plant Cell 9:1197-1210). Recently, another class of plant growthregulators, named brassinosteroids, has been identified that alsodramatically promote plant growth (Yokota, T. (1997) Trends Plant Sci.2:137-143; Azpiroz et al. (1998) Plant Cell 10:219-230; Choe et al.(1998) Plant Cell 10:231-243). However, the mechanisms by which planthormones act, either singly or in concert, to control cell elongationremains unclear.

[0008] One way to gain an understanding of mechanisms that mediate cellelongation is to study mutants in which this aspect of plant growth iscompromised (Klee et al. (1991) Annu. Rev. Plant Physiol. Plant Mol.Biol. 42:529-551). Numerous such mutants have been identified acrossmost plant species, including maize, in which more than 25 single-genemutations that affect plant stature have been characterized (Coe et al.(1988) In: Corn & Corn Improvement, G. F. Sprague (Ed.) Madison, Wis.;Sheridan, W. F. (1988) Annu. Rev. Genet. 22:353-385). These dwarfmutants are considered to be GA related, mainly because GA is the onlyphytohormone whose role in regulating height in maize has beenconvincingly established (Phinney et al. (1985) Curr. Top. PlantBiochem. Physiol. 4:67-74; Fujioka et al. (1988) Proc. Natl. Acad. Sci.USA 85:9031-9035). Both types of mutants, GA responsive and GAnon-responsive, have been found in this collection of maize mutants.While genes for a number of GA-responsive mutants have been cloned andfound to be involved in GA biosynthesis (Bensen et al. (1995) Plant Cell7:75-84; Winkler et al. (1995) Plant Cell 7:1307-1317), nothing is knownabout the nature of defects in GA non-responsive maize mutants.

[0009] One type of GA non-responsive dwarf mutants that have receivedmuch attention from maize geneticists and breeders is called brachytic.These dwarfs are characterized by internodes of substantially reducedlength, relative to wild-type, without having any effect on the size ornumber of other organs, including the leaves, ear and tassel (Kempton,J. H. (1920) J Hered. 11: 111-115). There are three known brachyticmutations in maize, br1, br2 and br3, all of which are recessive (Coe etal. (1988) In: Corn & Corn Improvement, G. F. Sprague (Ed.) Madison,Wis.; Sheridan, W. F. (1988) Annu. Rev. Genet. 22:353-385). Because ofthe commercial interest in br2 for enhancing plant productivity(Pendleton et al. (1961) Crop Sci. 1:433-435; Duvick, D. N. (1977)Maydica 22:187-196; Djisbar et al. (1987) Maydica 32:107-123; Russel, W.A. (1991) Adv. Agron. 46:245-298), this dwarf has been characterized themost. Depending on the genetic background, plants homozygous recessivefor br2 are 30-70% shorter than their normal sibs. This reduction inplant height is exclusively due to a reduction of the length of stalk(stem) internodes. In addition to being dwarf, br2 mutants grown undergreenhouse conditions often suffer from buggy whip, a disease-likecondition in which the unfurling leaves in the whorl undergo necrosisand stay stuck together. This condition often results in the death ofthe growing tip of the plant.

[0010] Although the dwarfing trait in maize has been extensively studiedboth genetically and molecularly, it has yet to be exploitedsuccessfully in breeding efforts in this crop plant. In contrast, dwarfmutants of sorghum have contributed significantly to the development ofmodern day cultivars. Sorghum and maize are both members of the grass(Poaceae or Gramineae) family and thus share many characteristicsincluding genomic organization and plant body form. Out of the fourdwarfing mutations exploited in sorghum, dw3, whose dwarfing phenotypelooks very similar to that of br2 in maize, appears to be the mostprominent. However, the only dw3 allele (dw3-rej) available thus far hasa serious problem which limits its agronomic value. The dwarf phenotypeassociated with the dw3 allele is unstable, with a reversion frequencyto wild-type (tall) as high as about 1% in certain genetic backgrounds.The instability of this dwarf phenotype, the mechanism of which haseluded sorghum geneticists thus far, not only continues to embarrasssorghum breeders, but also sometimes leads to the rejection of anotherwise promising inbred or hybrid.

[0011] To keep up with the demand for increased agricultural production,new targets are needed for genetically engineering agricultural plantsfor the improvement of agronomic characteristics. Elucidating themolecular mechanisms of cell division and elongation will provide newtargets for agricultural scientists to manipulate.

SUMMARY OF THE INVENTION

[0012] Compositions and methods for expressing genes encodingP-glycoproteins in plants are provided. The compositions comprisenucleotide sequences encoding P-glycoproteins, particularlyP-glycoproteins that control plant growth. The compositions furthercomprise nucleotide sequences of the Dw3 gene of sorghum. The sequencesof the invention are useful in transforming plants for tissue-preferredor constitutive expression of P-glycoproteins and for isolatinghomologous nucleotide molecules that encode P-glycoproteins. Suchsequences find use in methods for controlling the growth of organisms,particularly stem growth in plants. The sequences of the invention alsofind use in methods of enhancing the resistance of plants to pathogens.

[0013] The invention further encompasses methods for isolatingnucleotide molecules that are capable of controlling the growth ofplants. Such methods find use in the isolation of genes involved inplant growth processes.

[0014] Methods are provided for identifying plants that possess a mutantallele that is capable of conferring a stable mutant phenotype on anorganism. Such methods find use in agriculture, particularly in thebreeding of dwarf crop plants, particularly dwarf sorghum plants.

[0015] Expression cassettes comprising the sequences of the inventionare provided. Additionally provided are transformed plants, planttissues, plant cells and seeds thereof. Isolated proteins encoded by thenucleotide sequences of the invention are provided.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention is drawn to compositions and methods formanipulating the growth of organisms. The methods involve transformingorganisms with nucleotide sequences encoding P-glycoproteins. Inparticular, the nucleotide sequences are useful for controlling stemgrowth in plants. Thus, transformed plants, plant cells, plant tissuesand seeds are provided. Compositions are nucleic acids and proteinsrelating to P-glycoprotein or P-glycoprotein-like genes in plants. Moreparticularly, nucleotide sequences of the Dw3 gene of sorghum and theamino acid sequences of the proteins encoded thereby are disclosed. Thesequences find use in the construction of expression vectors forsubsequent transformation into plants of interest, as probes for theisolation of other P-glycoprotein-like genes, as molecular markers, andthe like.

[0017] The present invention discloses the first unequivocal evidence ofthe involvement of multidrug-resistance-like P-glycoproteins in thecontrol of growth and development in an organism. Thus, it is recognizedthat any P-glycoprotein known in the art that affects growth anddevelopment can be used in the practice of the invention. For example,five other plant P-glycoproteins are known. See, for example Dudler etal. (1998) Methods Enzym. 292:162-173 (Arabidopsis), Davies et al.(1997) Gene 199:195-202 (Barley), Wang et al. (1996) Plant Mol. Biol.31:683-687 (Potato) and GenBank Acession Numbers Y10227 and Y15990 (bothfrom Arabidopsis); herein incorporated by reference. These and otherP-glycoprotein sequences may be tested for an effect on growth bymethods such as, for example, transformation with antisense sequencesand monitoring effects on progeny plants.

[0018] The present invention also discloses methods for identifyinggenes encoding multidrug-resistance-like P-glycoproteins that controlthe growth of an organism, particularly a plant. An example of theidentification of such a gene is disclosed for the Dw3 gene of sorghum.Also provided is a method for identifying an allele of a gene whereinthe allele confers a stable dwarf phenotype on a plant. An embodiment ofthis method involves identifying stable mutant alleles of the Dw3 genethat confer a dwarf phenotype on sorghum plants.

[0019] Compositions of the invention include the native nucleotidesequences for P-glycoprotein genes, antisense sequences, as well asvariants and fragments thereof. Particularly, the P-glycoprotein gene ofthe sorghum Dw3 locus and the respective amino acid sequence for theP-glycoproteins encoded thereby, as well as fragments and variantsthereof are provided. The Dw3 nucleotide sequences are set forth in SEQID NOS: 1-3 and 7-8. The nucleotide sequences or corresponding antisensesequences find use in modulating the expression of a P-glycoprotein in aplant or plant cell. That is, the coding sequences can be used toincrease the expression while antisense sequences can be used todecrease expression.

[0020] The sequences of the invention find use in methods of modifyingthe growth of an organism. In an embodiment of the invention, nucleotidesequences of the invention find use in methods of modifying plantgrowth. Toward this end, the sequences of the invention may be utilizedin expression cassettes or nucleotide constructs operably linked to anyone of a variety of plant promoters. Aspects of plant growth that may beimpacted by the methods of the invention include, but are not limitedto, plant height; the size, shape and number of cells and organs; celldivision rate; cell elongation rate; the growth rate of the plant, itsorgans, tissues and cells; timing and location of organ initiation; lifespan; and the like.

[0021] The invention discloses methods for reducing plant growth whichfind use as alternatives to applying synthetic, growth-retardingchemicals to plants. These methods provide environmentally safealternatives to traditional means of retarding stem elongation or growthwith synthetic chemicals. Some embodiments of the invention make use ofplants transformed with tissue-preferred promoters, particularlystem-preferred promoters, operably linked to nucleotide sequencesencoding P-glycoproteins.

[0022] Methods are provided for reducing the growth of a plant. Suchmethods involve transforming plants with at least one nucleotidesequence of the invention. The nucleotide sequences may be used ineither the sense or antisense orientation to suppress the level of anendogenous P-glycoprotein that controls the growth of a plant. Byreducing the level in a plant of such a P-glycoprotein, particularly onethat controls stem or stalk growth, a plant of reduced stature, a dwarfplant, may be achieved. Dwarf plants having improved agronomiccharacteristics can be obtained by these methods. Such improvedagronomic characteristics include, but are not limited to, reducedpotential for lodging, increased water-use efficiency, reduced lifecycle, increased harvest efficiency and increased yield per unit area.The methods of the invention can eliminate the need to graft shoots offruit trees on dwarfing rootstocks to produce dwarf fruit trees.

[0023] The methods of the invention find use in producing dwarfvarieties of crop plants. In one embodiment of the invention, a dwarfBasmati rice plant is produced by transforming the plant with anucleotide sequence encoding at least a portion of a P-glycoprotein thatcontrols the growth of a plant. Basmati rice, known for its aromaticfragrance, slender, elongated grains, and relatively short cooking time,is the favorite type of rice of the majority of people in the Indiansub-continent. While commercially acceptable dwarf cultivars have beendeveloped for other types of rice, previous attempts to producecommercially acceptable varieties of Basmati rice by traditional plantbreeding methods have failed. While dwarf plants were obtained in suchattempts, some of the distinctive grain characteristics that consumersexpect in Basmati rice were not retained in the dwarf plants. Themethods of the invention provide a means of making dwarf Basmati riceplants that produce grain possessing the characteristics desired byconsumers.

[0024] The desired dwarf Basmati rice plants are produced bytransforming a non-dwarf Basmati rice plant with a nucleotide sequenceof the invention operably linked to a promoter that drives expression ina plant. While the choice of promoter depends on the desired outcome,the preferred promoters are tissue-preferred promoters, particularlystem-preferred promoters. Through cosuppression (sense suppression) orantisense suppression, such plants produce reduced levels of at leastone P-glycoprotein that controls the growth of the Basmati rice plant,particularly stem growth. Preferably, the nucleotide sequence encodes atleast a portion of a P-glycoprotein that controls the growth of a plant.More preferably, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOS: 1-3 and 7-8 or a nucleotide sequence thatencodes the amino acid sequence set forth in SEQ ID NOS: 4 or 9. Mostpreferably, the nucleotide sequence is from a rice gene that ishomologous to the sorghum gene, Dw3. Such a rice gene encodes aP-glycoprotein that that controls the growth of the stem of the riceplant. The methods of the invention comprise transforming plants withthe full-length nucleotide sequences of the invention, or any fragmentor part thereof.

[0025] Methods for enhancing the resistance of plants to pathogens areprovided. It is recognized that P-glycoproteins are involved inresistance mechanisms against pathogens. A mutant strain of thenematode, Caenorhabditis elegans, with deletions of two P-glycoproteingenes is substantially more susceptible to death than wild-typenematodes, when placed on a lawn of a Pseudomonas aeruginosa strain thatis a pathogen of both plants and animals (Mahajan-Miklos et al. (1999)Cell 96:47-56). Br2 is a maize gene that encodes amultidrug-resistance-like P-glycoprotein that controls plant growth,particularly stem growth (See U.S. Provisional Application Serial No.60/164,886 entitled “Genes and Methods for Manipulation of Growth” filedNov. 12, 1999; herein incorporated by reference). Maize plants that arehomozygous for the mutant allele, br2, display a dwarf stature, andunder certain cultural conditions, can also display a phenotype known as“buggy whip” which mimics a bacterial pathogen-induced necrosis of thegrowing tip of a plant.

[0026] The methods for the enhancing resistance of plants to pathogenscomprise transforming plants with the nucleotide sequences of theinvention operably linked to promoters that drive expression in a plant.Such plants display enhanced resistance to pathogens, includingbacteria, fungi, viruses, nematodes and insects. The methods find use inagriculture for limiting the impact of plant pathogens on cropproduction and provide an alternative to the use of synthetic pesticidesin controlling plant pathogens.

[0027] Also provided are methods for identifying a plant with a stablemutant phenotype. Such methods find use in agriculture, particularly inthe development of improved crop plants. The methods relate to aninsertion-induced, mutant phenotype. By “insertion-induced, mutantphenotype” is intended a mutant phenotype that is due to the insertionof a nucleotide, or a sequence of nucleotides, into the sequence of agene of interest. While the invention does not depend upon a particulargenetic mechanism for such an insertion-induced mutant phenotype, thepresence of such an insertion within a gene typically disrupts thenormal wild-type function of the gene, or gene product thereof. Whilethe methods of the invention are not bound by any particular type ofinsertion, such an insertion may be due to, for example, the insertionof a transposon or transposable element, or the duplication of anucleotide sequence such as those which are known to occur as a resultof genetic recombination.

[0028] Preferably, such an insertion-induced phenotype is unstable fromone generation to the next. That is, self pollination of one or morelike plants having the insertion-induced phenotype results in at leastone individual from among the resulting progeny population that hasreverted to the wild-type phenotype. More preferably, such phenotypicinstability, from one generation to the next, is due to the loss of atleast a portion of the insertion from the gene of interest and that sucha loss results in at least one progeny plant, which has reverted to awild-type phenotype. The methods of the present invention involveidentifying an individual with a stable mutant phenotype from among suchprogeny population.

[0029] To identify a plant possessing an allele of a gene that confers astable mutant phenotype, genomic DNA from a mutant plant is analyzed todetermine if at least one copy of the gene of interest lacks theinsertion, or at least a portion thereof. Generally, the mutant plant isselected from a population of progeny derived from the self pollinationof one or more plants having the insertion-induced, mutant phenotype.Typically, in a population of such progeny, wild-type revertants willalso be observed, indicating that at least a portion of the insertionhas excised from the gene of interest. The genomic DNA of the selectedmutant plant can be isolated and analyzed for the absence of all or aportion of the insertion by techniques known to those of ordinary skillin the art such as, for example, Southern blotting, restriction fragmentlength polymorphism (RFLP) analysis and DNA amplification by polymerasechain reaction (PCR). Once a mutant plant lacking a portion of theinsertion is identified, the progeny of such a mutant plant can bemonitored to verify phenotypic stability. If desired, subsequentgenerations can also be monitored.

[0030] Also provided are plants having stable mutant phenotypes andnucleotide sequences of alleles of genes which are capable of conferringa stable mutant phenotype on a plant.

[0031] A method of the invention involves identifying a sorghum plantwith a stable dwarf phenotype. Such a sorghum plant possesses in itsgenome a stable mutant allele of the Dw3 gene. Such a stable mutantallele is capable of conferring a stable dwarf phenotype on a sorghumplant and the nucleotide sequence of a fragment of such an allele is setforth in SEQ ID NO: 2. One method of the invention employs RFLP analysisutilizing Southern blotting with a probe derived from nucleotidesequences of maize Br2. This method additionally involves PCRamplification and DNA sequence analysis to determine the nucleotidesequence of the stable mutant allele.

[0032] Methods are provided for identifying nucleotide sequencesencoding gene products that control plant growth. Such gene products,like the DW3 protein, impact or, modify the growth of a plant indetectable way by, for example, affecting characteristics such as theheight or shape of a cell, organ or the plant body itself, cell number,cell division rate or cell elongation rate, organ growth rate,appearance of reproductive structures, timing and location of organinitiation and the like. The methods of the invention are particularlydirected toward nucleotide sequences which influence the height orstature of a plant. The nucleotide sequences of the invention find usein any method known to those skilled in the art for identifyinghomologous sequences. Such methods for identifying homologous sequencesinclude PCR amplification, hybridization, Southern blotting, colonyhybridization and the like.

[0033] An embodiment of the invention involves the use of PCRamplification to identify nucleotide sequences encoding gene productsthat control plant growth. Such PCR amplification comprises the use ofat least one oligonucleotide primer derived from a nucleotide sequenceencoding of a gene encoding a multidrug-resistance-like P-glycoprotein.Preferably, such a nucleotide sequence is from a gene that encodes aP-glycoprotein that controls the growth of an organism, particularly aplant. More preferably, the nucleotide sequence is selected from thegroup consisting of SEQ ID NOS: 1-3 and 7-8.

[0034] In another embodiment, oligonucleotide primers (SEQ ID NOS: 5-6)were prepared from the sequences of Br2. Such primers were used to PCRamplify Dw3 from genomic DNA isolated from sorghum plants. Following DNAsequencing the identity of Dw3 was revealed. In a similar manner, otherhomologues of both Br2 and Dw3 can be identified using the same primersor other primers derived from any gene encoding a P-glycoprotein thatcontrols the growth of an organism.

[0035] In still another exemplary embodiment of the invention, one ormore nucleotide sequences set forth in SEQ ID NOS: 1-3 and 5-8 or anucleotide sequence encoding the amino acid sequence set forth in SEQID. NO. 4 or 9 are used to design hybridization probes or PCR primers toidentify a gene in the genome of a Basmati rice plant that is homologousto the sorghum gene, Dw3. Preferably, such a gene, from a Basmati riceplant, encodes a P-glycoprotein. More preferably, such a gene encodes aP-glycoprotein that controls the growth of the Basmati rice plant. Mostpreferably, such a gene encodes a P-glycoprotein that controls the stemgrowth of the Basmati rice plant.

[0036] The P-glycoproteins of the invention encompass all polypeptidesand nucleotide sequences encoding them that share substantial sequenceidentity to the sequences of the invention whether or not suchpolypeptides possess covalently attached carbohydrates orcarbohydrate-containing chains.

[0037] By “control growth of an organism” is intended to includeimpacting, modifying, modulating, affecting, increasing, and decreasinggrowth and growth-related processes of an organism. Such processes mayinfluence any of a multitude of characteristics of an organismincluding, but not limited to, cell size and shape, organism size andshape, cell division rate, cell enlargement rate, organ growth rate,onset of reproductive maturity and life span.

[0038] By “mutant phenotype” is intended any non-wild-type, non-typicalor non-standard phenotype which occurs as a result of a geneticalteration in the genome of an organism. When used in reference todomesticated plants and animals, a “mutant phenotype” is any phenotypethat is substantially different from the typical phenotype of theparticular domesticated breed or cultivated variety from which themutant phenotype arose.

[0039] By “mutant plant” is intended a plant having a mutant phenotype.

[0040] By “mutant allele” is intended an allele of a gene that iscapable of causing a “mutant phenotype.”

[0041] By “dwarf” is intended a typically small. By “dwarf plant” isintended an a typically small plant. Generally, such a “dwarf plant” hasa stature or height that is reduced from that of a typical plant byabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% orgreater. Generally, but not exclusively, such a dwarf plant ischaracterized by a reduced stem, stalk or trunk length when compared tothe typical plant.

[0042] By “nucleotide molecule” is intended a molecule composed ofnucleotides covalently bound to one another. Nucleotides include bothribonucleotides and deoxyribonucleotides. “Nucleotide molecule”encompasses single-stranded and double stranded forms of both DNA andRNA. “Nucleotide molecules” may be naturally occurring, synthetic or acombination of both. The linear arrangement of nucleotides in a“nucleotide molecule” is referred to as a “nucleotide sequence” andunless specified otherwise is presented herein from left to rightcorresponding to 5′-to-3′ direction. Because of the complementary natureof the opposite strands of a double-stranded nucleotide molecule, anucleotide sequence of the invention additionally encompasses itscomplementary antisense sequence.

[0043] Compositions of the invention include native nucleotide sequencesfor genes encoding multidrug-resistance-like-gene-encodedP-glycoproteins, homologues of multidrug-resistance-like-gene-encodedP-glycoproteins, antisense sequences, as well as fragments and variantsand fragments thereof. In particular, the present invention provides forisolated nucleic acid molecules comprising nucleotide sequences encodingthe amino acid sequences shown in SEQ ID NOS: 4 and 9, or the nucleotidesequences encoding the DNA sequences deposited in a bacterial host asPatent Deposit No. PTA 2645. Further provided are polypeptides having anamino acid sequence encoded by a nucleic acid molecule described herein,for example those set forth in SEQ ID NOS: 3 and 8, respectively, thosedeposited in a bacterial host as Patent Deposit Nos. PTA 2645, andfragments and variants thereof.

[0044] Plasmids containing the nucleotide sequences of the inventionwere deposited with the Patent Depository of the American Type CultureCollection (ATCC), Manassas, Va., on Nov. 1, 2000 and assigned PatentDeposit No PTA 2645. These deposits will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. These depositswere made merely as a convenience for those of skill in the art and arenot an admission that a deposit is required under 35 U.S.C. §112.

[0045] The invention encompasses isolated or substantially purifiednucleic acid or protein compositions. An “isolated” or “purified”nucleic acid molecule or protein, or biologically active portionthereof, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Preferably, an “isolated” nucleic acid is free of sequences (preferablyprotein encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Aprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5%, (bydry weight) of contaminating protein. When the protein of the inventionor biologically active portion thereof is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals.

[0046] Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain biological activity of the native P-glycoprotein and hence retainone or more functions of the native P-glycoprotein such as, for example,transmembrane transporter activity and ATP binding. Alternatively,fragments of a nucleotide sequence that are useful as hybridizationprobes may or may not encode protein fragments retaining biologicalactivity. Thus, fragments of a nucleotide sequence may range from atleast about 20 nucleotides, about 50 nucleotides, about 100 nucleotides,and up to the full-length nucleotide sequence of the invention.

[0047] A fragment of a P-glycoprotein gene nucleotide sequence thatencodes a biologically active portion of a P-glycoprotein of theinvention will encode at least 15, 20, 25, 30, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000,1,100, 1,200, 1,300, or 1,400 contiguous amino acids, or up to the totalnumber of amino acids present in a full-length P-glycoprotein of theinvention (for example, 415 and 1,421 amino acids for SEQ ID NOS: 4 and9). Fragments of a P-glycoprotein gene nucleotide sequence that areuseful as hybridization probes for PCR primers generally need not encodea biologically active portion of a P-glycoprotein.

[0048] Thus, a fragment of a P-glycoprotein gene nucleotide sequence mayencode a biologically active portion of a P-glycoprotein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of aP-glycoprotein can be prepared by isolating a portion of one of theP-glycoprotein gene nucleotide sequences of the invention, expressingthe encoded portion of the P-glycoprotein e.g., by recombinantexpression in vitro), and assessing the activity of the portion of theP-glycoprotein. Nucleic acid molecules that are fragments of aP-glycoprotein gene nucleotide sequence comprise at least 16, 20, 50,75, 100, 150, 200, 300, 500, 700, 1,000, 1,200, 1,500, 2,000, 2,500,3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000 nucleotides, or up tothe number of nucleotides present in a full-length P-glycoproteinnucleotide sequence disclosed herein (for example, 2,139, 1,267, 1,261,6,827, and 4213 nucleotides for SEQ ID NOS: 1-3, and 7-8, respectively).

[0049] By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the P-glycoprotein polypeptides of theinvention. Naturally occurring allelic variants such as these can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode aP-glycoprotein protein of the invention. Generally, variants of aparticular nucleotide sequence of the invention will have at least about40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%,preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, andmore preferably at least about 98%, 99% or more sequence identity tothat particular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein using default parameters.

[0050] By “variant” protein is intended a protein derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Variant proteins encompassedby the present invention are biologically active, that is they continueto possess the desired biological activity of the native protein, thatis, transporter activity or ATP binding activity as described herein.Such variants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a nativeP-glycoprotein of the invention will have at least about 40%, 50%, 60%,65%, 70%, generally at least about 75%, 80%, 85%, preferably at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably atleast about 98%, 99% or more sequence identity to the amino acidsequence for the native protein as determined by sequence alignmentprograms described elsewhere herein using default parameters. Abiologically active variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

[0051] The proteins of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the P-glycoproteinscan be prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192;Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al. (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may bepreferred.

[0052] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the proteins of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants will continue to possess the desired transporter activity.Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and preferablywill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444.

[0053] The deletions, insertions, and substitutions of the proteinsequences encompassed herein are not expected to produce radical changesin the characteristics of the protein. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays.

[0054] Variant nucleotide sequences and proteins also encompassnucleotide sequences and proteins derived from a mutagenic andrecombinogenic procedure such as DNA shuffling. With such a procedure,one or more different P-glycoprotein coding sequences can be manipulatedto create a variant nucleotide sequence encoding a variantP-glycoprotein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the P-glycoproteingene of the invention and other known P-glycoprotein genes to obtain anew gene coding for a protein with an improved property of interest,such as an increased Km in the case of an enzyme. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0055] The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire sequences setforth herein or to fragments thereof are encompassed by the presentinvention. Such sequences include sequences that are orthologs of thedisclosed sequences. By “orthologs” is intended genes derived from acommon ancestral gene and which are found in different species as aresult of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encoded proteinsequences share substantial identity as defined elsewhere herein.Functions of orthologs are often highly conserved among species.

[0056] In a PCR approach, oligonucleotide primers can be designed foruse in PCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Irmis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

[0057] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the P-glycoprotein gene nucleotide sequences of the invention.Methods for preparation of probes for hybridization and for constructionof cDNA and genomic libraries are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0058] For example, the entire Dw3 sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding P-glycoprotein gene sequences and messengerRNAs. To achieve specific hybridization under a variety of conditions,such probes include sequences that are unique among P-glycoprotein genesequences and are preferably at least about 10 nucleotides in length,and most preferably at least about 20 nucleotides in length. Such probesmay be used to amplify corresponding P-glycoprotein gene sequences froma chosen plant by PCR. This technique may be used to isolate additionalcoding sequences from a desired plant or as a diagnostic assay todetermine the presence of coding sequences in a plant. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

[0059] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least two-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0060] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1×to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5×to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. The duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

[0061] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (%form)-500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with >90% identity aresought, the T_(m) can be decreased 110° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 101° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0062] Thus, isolated sequences that encode for P-glycoproteins andwhich hybridize under stringent conditions to the to the P-glycoproteingene sequences disclosed herein, or to fragments thereof, areencompassed by the present invention. Such sequences will be at leastabout 70% to 75%, about 80% to 85%, and even 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more homologous with the disclosed sequences.That is, the sequence identity of sequences may range, sharing at leastabout 70% to 75%, about 80% to 85%, and even at least about 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity.

[0063] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity.”

[0064] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0065] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0066] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0067] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used.Alignment may also be performed manually by inspection.

[0068] Unless otherwise stated, sequence identity/similarity valuesprovided herein refer to the value obtained using GAP Version 10 usingthe following parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

[0069] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest

[0070] number of matched bases and the fewest gaps. It allows for theprovision of a gap creation penalty and a gap extension penalty in unitsof matched bases. GAP must make a profit of gap creation penalty numberof matches for each gap it inserts. If a gap extension penalty greaterthan zero is chosen, GAP must, in addition, make a profit for each gapinserted of the length of the gap times the gap extension penalty.Default gap creation penalty values and gap extension penalty values inVersion 10 of the Wisconsin Genetics Software Package for proteinsequences are 8 and 2, respectively. For nucleotide sequences thedefault gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

[0071] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0072] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference tothe residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0073] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0074] (e)(i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0075] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C. lower than the T_(m), depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

[0076] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Preferably,optimal alignment is conducted using the homology alignment algorithm ofNeedleman et al. (1970) J. Mol. Biol. 48:443. An indication that twopeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

[0077] The use of the term “nucleotide constructs” herein is notintended to limit the present invention to nucleotide constructscomprising DNA. Those of ordinary skill in the art will recognize thatnucleotide constructs, particularly polynucleotides andoligonucleotides, comprised of ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides may also be employed in themethods disclosed herein. Thus, the nucleotide constructs of the presentinvention encompass all nucleotide constructs that can be employed inthe methods of the present invention for transforming plants including,but not limited to, those comprised of deoxyribonucleotides,ribonucleotides, and combinations thereof. Such deoxyribonucleotides andribonucleotides include both naturally occurring molecules and syntheticanalogues. The nucleotide constructs of the invention also encompass allforms of nucleotide constructs including, but not limited to,single-stranded forms, double-stranded forms, hairpins, stem-and-loopstructures, and the like.

[0078] Furthermore, it is recognized that the methods of the inventionmay employ a nucleotide construct that is capable of directing, in atransformed plant, the expression of at least one protein, or at leastone RNA, such as, for example, an antisense RNA that is complementary toat least a portion of an mRNA. Typically such a nucleotide construct iscomprised of a coding sequence for a protein or an RNA operably linkedto 5′ and 3′ transcriptional regulatory regions. Alternatively, it isalso recognized that the methods of the invention may employ anucleotide construct that is not capable of directing, in a transformedplant, the expression of a protein or an RNA.

[0079] In addition, it is recognized that methods of the presentinvention do not depend on the incorporation into the genome of theentire nucleotide construct comprising a P-glycoprotein nucleotidesequence, only that the plant or cell thereof is altered as a result ofthe introduction of the nucleotide construct into a cell. In oneembodiment of the invention, the genome may be altered following theintroduction of the nucleotide construct into a cell. For example, thenucleotide construct, or any part thereof, may incorporate into thegenome of the plant. Alterations to the genome of the present inventioninclude, but are not limited to, additions, deletions, and substitutionsof nucleotides in the genome. While the methods of the present inventiondo not depend on additions, deletions, or substitutions of anyparticular number of nucleotides, it is recognized that such additions,deletions, or substitutions comprise at least one nucleotide.

[0080] The nucleotide constructs of the invention also encompassnucleotide constructs that may be employed in methods for altering ormutating a genomic nucleotide sequence in an organism, including, butnot limited to, chimeric vectors, chimeric mutational vectors, chimericrepair vectors, mixed-duplex oligonucleotides, self-complementarychimeric oligonucleotides, and recombinogenic oligonucleobases. Suchnucleotide constructs and methods of use, such as, for example,chimeraplasty, are known in the art. Chimeraplasty involves the use ofsuch nucleotide constructs to introduce site-specific changes into thesequence of genomic DNA within an organism. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984;all of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.

[0081] The invention encompasses the use of methods, such as, forexample, chimeraplasty to alter P-glycoprotein genes in plants. Suchalterations include, for example, changes in the coding sequence thatalter the amino acid sequence of the P-glycoprotein encoded thereby,resulting in a reduction in, or loss of, the function of theP-glycoprotein encoded by that gene.

[0082] The P-glycoprotein nucleotide sequences of the invention areprovided in expression cassettes for expression in the plant ofinterest. The cassette will include 5′- and 3′-regulatory sequencesoperably linked to a P-glycoprotein nucleotide sequence of theinvention. By “operably linked” is intended a functional linkage betweena promoter and a second sequence, wherein the promoter sequenceinitiates and mediates transcription of the DNA sequence correspondingto the second sequence. Generally, operably linked means that thenucleic acid sequences being linked are contiguous and, where necessaryto join two protein coding regions, contiguous and in the same readingframe. The cassette may additionally contain at least one additionalgene to be cotransformed into the organism. Alternatively, theadditional gene(s) can be provided on multiple expression cassettes.

[0083] Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the P-glycoprotein nucleotidesequence to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes.

[0084] The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aP-glycoprotein nucleotide sequence of the invention, and atranscriptional and translational termination region functional inplants. The transcriptional initiation region, the promoter, may benative or analogous or foreign or heterologous to the plant host.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. By “foreign” is intended that the transcriptionalinitiation region is not found in the native plant into which thetranscriptional initiation region is introduced.

[0085] While it may be preferable to express the sequences usingheterologous promoters, the native promoter sequences may be used. Suchconstructs would change expression levels of a P-glycoprotein in theplant or plant cell. Thus, the phenotype of the plant or plant cell isaltered.

[0086] The termination region may be native with the transcriptionalinitiation region, may be native with the operably linked DNA sequenceof interest, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0087] Where appropriate, the gene(s) may be optimized for increasedexpression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons for improved expression.Methods are available in the art for synthesizing plant-preferred genes.See, for example, U.S. Pat. No. 5,380,831, and 5,436,391, and Murray etal. (1989) Nucleic Acids Res. 17:477-498, herein incorporated byreference.

[0088] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. When possible, the sequence is modified to avoid predicted hairpinsecondary mRNA structures.

[0089] The expression cassettes may additionally contain 5′-leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′-noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Seealso, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

[0090] In preparing the expression cassette, the various DNA fragmentsmay be manipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

[0091] It is recognized that with the nucleotide sequences of theinvention, antisense constructions, complementary to at least a portionof the messenger RNA (mRNA) for the P-glycoprotein gene sequences can beconstructed. Antisense nucleotides are constructed to hybridize with thecorresponding mRNA. Modifications of the antisense sequences may be madeas long as the sequences hybridize to and interfere with expression ofthe corresponding mRNA. In this manner, antisense constructions having70%, preferably 80%, more preferably 85% sequence identity to thecorresponding target sequences may be used. Furthermore, portions of theantisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, or greater may be used.

[0092] The nucleotide sequences of the present invention may also beused in the sense orientation to suppress the expression of endogenousgenes in plants. Methods for suppressing gene expression in plants usingnucleotide sequences in the sense orientation, also known ascosuppression methods, are known in the art. The methods generallyinvolve transforming plants with a nucleotide construct comprising apromoter that drives expression in a plant operably linked to at least aportion of a nucleotide sequence that corresponds to the transcript ofthe endogenous gene. Typically, such a nucleotide sequence hassubstantial sequence identity to the sequence of the transcript of theendogenous gene, preferably greater than about 65% sequence identity,more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See, U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

[0093] Generally, the expression cassette will comprise a selectablemarker gene for the selection of transformed cells. Selectable markergenes are utilized for the selection of transformed cells or tissues.Marker genes include genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO) and hygromycinphosphotransferase (HPT), as well as genes conferring resistance toherbicidal compounds, such as glufosinate ammonium, bromoxynil,imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally,Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al.(1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al.(1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566;Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

[0094] The above list of selectable marker genes is not meant to belimiting. Any selectable marker gene can be used in the presentinvention.

[0095] A number of promoters can be used in the practice of theinvention. The promoters may be selected based on the desired timing,localization and level of expression of the P-glycoprotein genes in aplant. Constitutive, tissue-preferred, pathogen-inducible,wound-inducible and chemically regulatable promoters can be used in thepractice of the invention.

[0096] Such constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV ³⁵Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. application Ser. No. 08/409,297), and the like. Otherconstitutive promoters include, for example, U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and5,608,142.

[0097] Tissue-preferred promoters can be utilized to target enhancedP-glycoprotein expression within a particular plant tissue.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

[0098] Leaf-preferred promoters include, Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J 4(3):495-505.

[0099] Root-preferred promoters are known and can be selected from themany available from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2): 207-218 (soybean root-preferred glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-preferredcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-preferred promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-preferred promoters isolated fromhemoglobin genes from the nitrogen-fixing nonlegume Parasponiaandersonii and the related non-nitrogen-fixing nonlegume Trema tomentosaare described. The promoters of these genes were linked to aβ-glucuronidase reporter gene and introduced into both the nonlegumeNicotiana tabacum and the legume Lotus corniculatus, and in bothinstances root-preferred promoter activity was preserved. Leach andAoyagi (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see Plant Science (Limerick) 79(1):69-76). They concluded that enhancerand tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri et al. (1989) used gene fusion to lacZ to show that theAgrobacterium T-DNA gene encoding octopine synthase is especially activein the epidermis of the root tip and that the TR2′ gene is rootpreferred in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′gene, fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0100] Generally, it will be beneficial to express the gene from aninducible promoter, particularly from a pathogen-inducible promoter.Such promoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also the copending applications entitled“Inducible Maize Promoters”, U.S. Application Serial No. 60/076,100,filed Feb. 26, 1998, and U.S. Application Serial No. 60/079,648, filedMar. 27, 1998, both of which are herein incorporated by reference.

[0101] Of interest are promoters that are expressed locally at or nearthe site of pathogen infection. See, for example, Marineau et al. (1987)Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

[0102] Additionally, as pathogens find entry into plants through woundsor insect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIPI (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J 6(2):141-150); and the like,herein incorporated by reference.

[0103] Chemically regulated promoters can be used to modulate theexpression of a gene in a plant through the application of an exogenouschemical regulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemically induciblepromoters are known in the art and include, but are not limited to, themaize in2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemically regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J 14(2):247-257) and tetracycline-inducibleand tetracycline-repressible promoters (see, for example, Gatz et al.(1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and5,789,156), herein incorporated by reference.

[0104] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);and McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bytebier et al. (1987) Proc.Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) inThe Experimental Manipulation of Ovule Tissues, ed. Chapman et al.(Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant CellReports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

[0105] Alternatively, the nucleotide sequences of the invention can beintroduced into an organism and allowed to undergo recombination withhomologous regions of the organism's genome. Such homologousrecombination approaches are well known to those of ordinary skill inthe art and can be used to stably incorporate sequences of the inventioninto an organism. Further, such strategies can be used to introduce“knockout mutations” into a specific gene of an organism that sharessubstantial homology to the sequences of the invention. A knockoutmutation is any mutation in the sequence of a gene that eliminates orsubstantially reduces the function or the level of the product encodedby the gene. Methods involving transformation of an organism followed byhomologous recombination to stably integrate the sequences of theinvention into the genome organism are encompassed by the invention. Theinvention is particularly directed to methods where sequences of theinvention are utilized to alter the growth of an organism. Such methodsencompass use of the sequences of the invention to interfere with thefunction or synthesis of a P-glycoprotein that controls growth of anorganism.

[0106] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure constitutive expression of the desired phenotypiccharacteristic has been achieved.

[0107] The present invention may be used for transformation of any plantspecies, including, but not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

[0108] Vegetables include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolius vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Ak. yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, rice, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.), more preferably corn, rice and sorghum plants.

[0109] The invention is drawn to compositions and methods for increasingthe resistance of a plant to a pathogen. Accordingly, the compositionsand methods are also useful in protecting plants against fungalpathogens, viruses, nematodes, insects, acarids and the like.

[0110] By “disease resistance” is intended that the plants avoid thedisease symptoms that are the outcome of plant-pathogen interactions.That is, pathogens are prevented from causing plant diseases and theassociated disease symptoms, or alternatively, the disease symptomscaused by the pathogen is minimized or lessened. The methods of theinvention can be utilized to protect plants from disease, particularlythose diseases that are caused by plant pathogens.

[0111] By “antipathogenic compositions” is intended that thecompositions of the invention have antipathogenic activity and thus arecapable of suppressing, controlling, and/or killing the invadingpathogenic organism. An antipathogenic composition of the invention willreduce the disease symptoms resulting from pathogen challenge by atleast about 5% to about 50%, at least about 10% to about 60%, at leastabout 30% to about 70%, at least about 40% to about 80%, or at leastabout 50% to about 90% or greater. Hence, the methods of the inventioncan be utilized to protect plants from disease, particularly thosediseases that are caused by plant pathogens.

[0112] Assays that measure antipathogenic activity are commonly known inthe art, as are methods to quantitate disease resistance in plantsfollowing pathogen infection. See, for example, U.S. Pat. No. 5,614,395,herein incorporated by reference. Such techniques include, measuringover time, the average lesion diameter, the pathogen biomass, and theoverall percentage of decayed plant tissues. For example, a plant eitherexpressing an antipathogenic polypeptide or having an antipathogeniccomposition applied to its surface shows a decrease in tissue necrosis(i.e., lesion diameter) or a decrease in plant death following pathogenchallenge when compared to a control plant that was not exposed to theantipathogenic composition. Alternatively, antipathogenic activity canbe measured by a decrease in pathogen biomass. For example, a plantexpressing an antipathogenic polypeptide or exposed to an antipathogeniccomposition is challenged with a pathogen of interest. Over time, tissuesamples from the pathogen-inoculated tissues are obtained and RNA isextracted. The percent of a specific pathogen RNA transcript relative tothe level of a plant specific transcript allows the level of pathogenbiomass to be determined. See, for example, Thomma et al. (1998) PlantBiology 95:15107-15111, herein incorporated by reference.

[0113] Furthermore, in vitro antipathogenic assays include, for example,the addition of varying concentrations of the antipathogenic compositionto paper disks and placing the disks on agar containing a suspension ofthe pathogen of interest. Following incubation, clear inhibition zonesdevelop around the discs that contain an effective concentration of theantipathogenic polypeptide (Liu et al. (1994) Plant Biology91:1888-1892, herein incorporated by reference). Additionally,microspectrophotometrical analysis can be used to measure the in vitroantipathogenic properties of a composition (Hu et al. (1997) Plant Mol.Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233,both of which are herein incorporated by reference).

[0114] Pathogens of the invention include, but are not limited to,viruses or viroids, bacteria, insects, nematodes, fungi, and the like.Viruses include any plant virus, for example, tobacco or cucumber mosaicvirus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.Specific fungal and viral pathogens for the major crops include:Soybeans: Phytophthora megasperma fsp. glycinea, Macrophominaphaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusariumoxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium roltsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotichum truncatum), Corynespora cassiicola, Septoria glycines,Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v.glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,Phakopsora pachyrhizi, Pythium aphnmidermatum, Pythium ultimum, Pythiumdebaryanum, Tomato spotted wilt virus, Heterodera glycines Fusariumsolani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeriamaculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Alternaria alternata; Alfalfa: Clavibater michiganese subsp. insidiosum,Pythium ultimum, Pythium irregulare, Pythium splendens, Pythiumdebaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium,Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphyliumherbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p.v.atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata,Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum,Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporiumgramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum,Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides,Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum,Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus,Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat SpindleStreak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletiatritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctoniasolani, Pythium arrhenomannes, Pythium gramicola, Pythiumaphanidermatum, High Plains Virus, European wheat striate virus;Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum, AsterYellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi,Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophominaphaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus,Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwiniacarotovorum pv. carotovora, Cephalosporium acremonium, Phytophthoracryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var.subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae(Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythiumirregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens,Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolarismaydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I,II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganense subsp.nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora,Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v.syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconiacircinata, Fusarium moniliforme, Alternaria alternata, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas dvenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthonamacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,Pythium arrhenomanes, Pythium graminicola, etc.

[0115] Nematodes include parasitic nematodes such as root-knot, cyst,and lesion nematodes, including Heterodera and Globodera spp;particularly Globodera rostochiensis and globodera pailida (potato cystnematodes); Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); and Heterodera avenae (cereal cystnematode).

[0116] Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pestsof the invention for the major crops include: Maize: Ostrinia nubilalis,European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea,corn earworm; Spodoptera frugiperda, fall armyworm; Diatraeagrandiosella, southwestern corn borer; Elasmopalpus lignosellus, lessercornstalk borer; Diatraea saccharalis, surgarcane borer; Diabroticavirgifera, western corn rootworm; Diabrotica longicornis barberi,northern corn rootworm; Diabrotica undecimpunctata howardi, southerncorn rootworm; Melanotus spp., wireworms; Cyclocephala borealis,northern masked chafer (white grub); Cyclocephala immaculata, southernmasked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafininer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Siphaflava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall army worm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis grandis, boll weevil;Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cottonfleahopper; Trialeurodes abutilonea, banded winged whitefly; Lyguslineolaris, tarnished plant bug; Melanoplus femurrubrum, redleggedgrasshopper; Melanoplus differentialis, differential grasshopper; Thripstabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall army worm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

[0117] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLE 1 Sorghum Dwarfing Gene, Dw3, Encodes a P-Glycoprotein Homologue

[0118] It is well established that the sorghum dwarfing phenotypeconferred by the dw3 recessive mutation is unstable, although themechanism responsible for its instability remains unknown. The dw3allele, referred to here as the reference allele (dw3-ref), reverts backto the wild-type form (conferring a tall phenotype) with a frequency ofabout 0.4% to about 1%. As a result, it is a commonplace to witness anumber of tall sorghum plants in a field of dw3 dwarfs. To determine ifthere is any relationship between the maize br2 gene and the sorghum dw3gene, leaf samples were collected from 8 dwarf and 8 tall (revertant)plants; these were expected to be true dw3 isogenics (identicalthroughout the genome except at the dw3 locus). The DNA of these sampleswas extracted, digested with PstI, and subjected to Southern blotanalysis using a probe from the maize Br2 gene. A clear and consistentDNA polymorphism was observed between the tall and dwarf plants, withthe restriction fragment from the revertant allele being about 1.0 kbsmaller that the dw3-ref allele.

[0119] Two conclusions were made from this result. First, the sorghumDw3 locus is structurally and functionally homologous to the maize Br2gene, suggesting that they may turn out to be true orthologs (i.e.,derived from the same ancestral gene by vertical descent). Second, sinceall revertants had the same RFLP pattern, and that the size of therevertant allele was smaller than the mutant allele, the mutable dw3-refallele was probably caused by an insertion. To address the latterinterpretation, sorghum DNA in the vicinity of the Br2-detectedpolymorphism was subjected to PCR amplification using twooligonucleotide primers (SEQ ID NOS: 5-6) derived from the nucleotidesequence of the maize Br2 gene.

[0120] PCR products were amplified from genomic DNA isolated from thetall revertants and dwarf plants with the dw3-ref allele. The PCRproducts were subsequently cloned and sequenced. The results obtainedshowed that a duplication of 882 bp had occurred in exon 5 of the dw3gene that led to the generation of the dw3-ref mutant allele (SEQ ID NO:1). Thus, the dw3 dwarf phenotype in sorghum is likely due to aninsertion-induced mutation that occurred within the Dw3 allele to giverise to the dw3-ref allele. A partial sequence of the tall revertantallele, designated Dw3-T is disclosed in SEQ ID NO: 3. The duplicationpresent in the dw3-ref mutant allele also seems to be responsible forthe unstable nature of dw3-ref. By an undetermined mechanism, thisduplication is removed in tall revertants of dw3-ref.

[0121] Comparison of the partial amino acid sequence of the proteinencoded by Dw3-T (SEQ ID NO: 4) revealed that, like BR2, this proteinbelongs to the family of multidrug-resistance-like P-glycoproteins.Whereas it shows more than 96% amino acid identity with the maize pgp1(the Br2 gene), it exhibits 81% and 79% identity with P-glycoproteingenes of Arabidopsis thaliana and potato respectively.

[0122] Since the instability of the dw3-ref allele may result from somegenetic recombination between two copies of the duplicated part of thegene, it might not always be precise. Some instances may occur where oneor more extra base pairs may be left behind or deleted, leading ineither case to a frame shift mutation. Such events are thus expected togenerate new mutant alleles of dw3 that are devoid of the duplication.And since the duplication seems to be responsible for the instability ofdw3-ref, the new mutant alleles of dw3 are expected to exhibit a stabledwarfing phenotype. Such stable dwarf alleles are highly desirable forbreeding improved sorghum cultivars, as the instability of dw3 has beena constant nemesis for breeders for enhancing the production of sorghum.

[0123] To identify a stable dw3 allele, DNA was extracted from 200 dwarfsorghum plants and subjected to Southern blot analysis using a probefrom the maize Br2 gene. Two dwarf plants were identified that exhibiteda restriction pattern that was different from the rest of the dwarfplants. Genomic DNA was isolated from one of these two dwarf plants andamplified using the oligonucleotide primers (SEQ ID NOS: 5-6) asdescribed supra. The PCR product was cloned and sequenced. Comparison ofthe nucleotide sequence of the cloned PCR product (SEQ ID NO: 2) fromthis dwarf plant to the sequence of dw3-ref (SEQ ID NO: 1) revealed thatthe duplication present in dw-3-ref was lost. Thus, this dwarf plantpossesses a new dw3 allele, designated as dw3-1. Comparison of thenucleotide sequence of the dw3-1 allele with the Dw3-T alleledemonstrated that the new dw3-1 allele has undergone minor changes.

[0124] To separate the new dw3-1 allele from the parental dw3-refallele, the dwarf plant possessing the dw3-1 was self pollinated andseeds from plant were collected and planted. From the progeny, plantsthat were homozygous for dw3-1 were identified by Southern blotanalysis, and the homozygous plants are being propagated to developstable dwarfing germplasm for sorghum. In addition, eight separatePioneer proprietary sorghum inbreds are also being genotyped for thepresence of new mutant derivatives of dw3-ref. The inbreds that wereutilized are AGK1G, MK7G, MQC100G, ZYL24, YYU28W, CAJ14W, FYL14W, andYGC87W. They were selected on the basis of their reversion frequency,which was rated high, moderate or low. These inbreds were plantedoutdoors in Johnston, Iowa during the summer of 1999. Two hundred plantsfrom each line were RFLP genotyped by digesting their DNA with PstI andhybridizing the resulting blots with a gene specific probe from the 3′end of the maize br2 gene. Four stable homozygous dwarf plants wereidentified from YYU28W and ten such plants were identified from FYL14W.Seeds from these stable dwarf plants have been harvested. The progeny ofthese stable dwarf plants can be used directly for the production ofhigh-yielding sorghum hybrids with the desired stable dwarf phenotype.

EXAMPLE 2 Nucleotide Sequence of a Dw3 Gene that Encodes a FunctionalGene Product

[0125] In order to clone the entire sequence from both the functional(Dw3) and the mutant (dw3-ref) alleles of the dw3 locus, a tallrevertant plant and a dwarf sibling were selected from the inbred AGK1G.In this inbred line, tall plants appear at a frequency of 0.1-0.4%. Thegenotype of the tall revertant plant was expected to be heterozygous atthe dw3 locus but otherwise identical to its dwarf sibling throughoutthe genome. To confirm that the tall revertant was heterozygous at thedw3 locus, DNA samples isolated from this plant and a number of dwarfsiblings were characterized by Southern analysis using three probesrepresenting the 5′, middle and 3′ parts of the maize br2 gene. Asexpected, polymorphism between dwarf siblings and the tall plant waslocalized only at the 3′ end of dw3. This analysis allowed theidentification of two EcoRI fragments from the tall revertant that whencombined contained the entire Dw3 allele. These were a 14 kb EcoRIfragment that contained the 5′ portion of the gene and an 8.1 kbfragment that contained the rest of the gene. A 9.0 kb fragment from thedw3-ref allele was determined to correspond to the 8.1 kb EcoRI fragmentof the Dw3 allele. Three size-selected libraries (containing the 14kb/EcoRI and 8.1 kb/EcoRI fragments from the tall revertant and the 9.0kb/EcoRI fragment from the dwarf sibling) were constructed in Lambdacloning vectors of Stratagene. The 14.0 kb/EcoRI fragment library wasconstructed in X Dash II and was screened with a probe coming from theextreme 5′ end of the maize br2 gene. The other two libraries wereprepared in X ZapII and were screened with a probe from the 3′end of themaize br2 gene. Positive clones were isolated and X DNA was extractedfor each of these clones. The Dw3 and dw3 genes were PCR amplified intofour overlapping 0.5 kb, 2.4 kb, 3.0 kb, and 1:3 kb fragments using genespecific primers and X DNA as a template. These PCR fragments werecloned in TOPO vector (Invitrogen). From the dwarf dw3 clone of 9.0 kb,a unique 888 bp SacI fragment containing a part of the duplicated regionwas subcloned into pBSK+vector (Stratagene).

[0126] DNA from at least two colonies of each PCR clone was sequencedusing Ml 3 forward, M13 reverse, and gene specific primers (GSPs). The888 bp SacI fragment from the dw3-ref clone was sequenced by using T3and T7 vector-specific primers alone. Sequence information, both fromthe extreme 5′ and 3′ ends of Dw3 and dw3 genes, was gathered bysequencing directly the X DNA of both the 14.0 kb and 8.1 kb clones,using gene-specific primers. All of the sequence information wascompiled and compared to reveal the cause of dwarfing in sorghum. Apairwise alignments between Dw3 and Br2 genes was done at the proteinlevel by using Clustal W Program and at the nucleotide level by usingBLAST Program of NCBL.

[0127] A polynucleotide of 6827 bp containing the full length Dw3 genewas assembled and is presented in SEQ ID NO: 7. Structurally, the Dw3gene has five exons and four introns. The length of five exons, fromexon 1 through exon 5, is 616 bp, 537 bp, 326 bp, 230 bp, and 2400 bp,respectively. Intron 1 is 165 bp (nucleotides 639-803 of SEQ ID NO: 7);intron 2 is 110 bp (nucleotides 1441-1550); intron 3 is 846 bp(nucleotides 1877-2722); and intron 4 is 1471 bp (nucleotides 2953-4423)in length. The intron/exon boundaries of Dw3 are identical to that ofthe br2 gene of maize. The predicted Dw3-cDNA is 4209 bp long from thestart codon to the end of the termination codon (SEQ ID NO: 8) and isthus 28 bp longer than the analogous region of the Br2-cDNA. Similarly,the predicted protein encoded by Dw3 is 1402 amino acids long (SEQ IDNO: 9), as compared to the 1394 amino acids predicted protein from Br2gene. Multiple alignment results show that overall Dw3 is 92% and 91.8%identical to the maize Br2 gene at the nucleotide level and at the aminoacid level, respectively.

[0128] PCR analysis of the polymorphic region between dw3 and br2 hadearlier suggested that a duplication of a part of exon 5 resulted in thedw3-ref dwarfing allelle of sorghum. To address if it was exclusivelythe reason for the mutant nature of the dw3-ref allele, the sequence ofthe Dw3 allele from the tall revertant was compared with that of thedw3-ref allele. As shown previously, the difference was detected only inexon 5 between these alleles. In the mutant allele (dw3-ref, SEQ ID NO:1), a stretch of 882 bp in exon 5 (from nucleotides 5650-6531 of SEQ IDNO: 7) is duplicated at the 6532 nucleotide position in the samedirection. This duplication converted the 1312 bp PstI restrictionfragment (from nucleotides 5463 bp to 6775 of SEQ ID NO: 7) in thefunctional Dw3 allele to the 2194 bp PstI fragment found in the dw3-refallele, and thus was the cause for the polymorphism between these twoalleles. Since no other changes were found between these alleles, theresults clearly implicate this duplication as the sole cause forcreating the dw3 dwarfing allele of sorghum. The addition of 882 bp tothe cDNA will no doubt have a serious ramification for the structure andactivity of the DW3 protein. These findings also show how the dw3-refallele spontaneously corrects itself, every now and then, by getting ridof the duplication. The mechanism, by which this correction occurs,remains unknown, as does the mechanism by which the duplication occurredin the first place.

Example 3 Transformation of Maize by Particle Bombardment andRegeneration of Transgenic Plants

[0129] Immature maize embryos from greenhouse donor plants are bombardedwith a plasmid containing a P-glycoprotein nucleotide sequence of theinvention operably linked to a promoter that drives expression in aplant and the selectable marker gene PAT (Wohlleben et al. (1988) Gene70:25-37), which confers resistance to the herbicide Bialaphos.Alternatively, the selectable marker gene is provided on a separateplasmid. Transformation is performed as follows. Media recipes followbelow.

[0130] Preparation of Target Tissue

[0131] The ears are husked and surface sterilized in 30% Clorox bleachplus 0.5% Micro detergent for 20 minutes, and rinsed two times withsterile water. The immature embryos are excised and placed embryo axisside down (scutellum side up), 25 embryos per plate, on 560Y medium for4 hours and then aligned within the 2.5-cm target zone in preparationfor bombardment.

[0132] Preparation of DNA

[0133] A plasmid vector comprising the P-glycoprotein nucleotidesequence of the invention operably linked to the plant promoter ofinterest is made. This plasmid DNA plus plasmid DNA containing a PATselectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows:

[0134] 100 μl prepared tungsten particles in water

[0135] 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

[0136] 100 μl 2.5 M CaCl₂

[0137] 10 μl 0.1 M spermidine

[0138] Each reagent is added sequentially to the tungsten particlesuspension, while maintained on the multitube vortexer. The finalmixture is sonicated briefly and allowed to incubate under constantvortexing for 10 minutes. After the precipitation period, the tubes arecentrifuged briefly, liquid removed, washed with 500 ml 100% ethanol,and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl100% ethanol is added to the final tungsten particle pellet. Forparticle gun bombardment, the tungsten/DNA particles are brieflysonicated and 10 μl spotted onto the center of each macrocarrier andallowed to dry about 2 minutes before bombardment.

[0139] Particle Gun Treatment

[0140] The sample plates are bombarded at level #4 in particle gun#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with atotal of ten aliquots taken from each tube of prepared particles/DNA.

[0141] Subsequent Treatment

[0142] Following bombardment, the embryos are kept on 560Y medium for 2days, then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for dwarf phenotype or other phenotypeassociated with expression of the P-glycoprotein nucleotides sequence ofthe invention.

[0143] Bombardment and Culture Media

[0144] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-1H₂O following adjustment to pH 5.8 with KOH);2.0 g/l Gelrite (added after bringing to volume with D-1H₂O); and 8.5mg/l silver nitrate (added after sterilizing the medium and cooling toroom temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-1H₂O following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-1H₂O); and 0.85 mg/lsilver nitrate and 3.0 mg/l bialaphos (both added after sterilizing themedium and cooling to room temperature).

[0145] Plant regeneration medium (288J) comprises 4.3 g/l MS salts(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 gnicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40g/l glycine brought to volume with polished D-1H₂O) (Murashige and Skoog(1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin,60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volumewith polished D-1H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-1H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-1H₂O), 0.1 g/l myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-1H₂O afteradjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing tovolume with polished D-1H₂O), sterilized and cooled to 60° C.

Example 4 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

[0146] For Agrobacterium-mediated transformation of maize with aP-glycoprotein nucleotide sequence of the invention, preferably themethod of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patentpublication WO98/32326; the contents of which are hereby incorporated byreference). Briefly, immature embryos are isolated from maize and theembryos contacted with a suspension of Agrobacterium, where the bacteriaare capable of transferring the P-glycoprotein nucleotide sequence ofthe invention to at least one cell of at least one of the immatureembryos (step 1: the infection step). In this step the immature embryosare preferably immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Preferably theimmature embryos are cultured on solid medium following the infectionstep. Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

[0147] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0148] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence having at least 90% identity to the sequence setforth in SEQ ID NO: 7; (b) a nucleotide sequence having at least 90%identity to the sequence set forth in SEQ ID NO: 8; (c) a nucleotidesequence that hybridizes under stringent conditions to at least onenucleotide sequence selected from the group consisting of the nucleotidesequence set forth in SEQ ID NO: 7 and the nucleotide sequence set forthSEQ ID NO: 8, said stringent conditions comprising hybridization at 37°C. in 50% formamide, 1 M NaCl, and 1% SDS and a wash in 0.1×SSC at 60 to65° C.; and (d) a nucleotide sequence that is fully complementary to anucleotide sequence selected from the group consisting of the nucleotidesequences set forth in (a)-(c); wherein said nucleotide molecule encodesa P-glycoprotein that controls plant growth or said nucleotide moleculeis complementary to a nucleotide sequence that encodes saidP-glycoprotein.
 2. An expression cassette comprising the nucleic acidmolecule of claim 1, said nucleotide sequence operably linked to apromoter that drives expression in a plant cell.
 3. The expressioncassette of claim 2, wherein said promoter is selected from the groupconsisting of tissue-preferred, constitutive, chemically regulatable,and pathogen-inducible promoters.
 4. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence having at least 95% identity to the sequenceset forth in SEQ ID NO: 7; (b) a nucleotide sequence having at least 95%identity to the sequence set forth in SEQ ID NO: 8; and (c) a nucleotidesequence that is fully complementary to a nucleotide sequence selectedfrom the group consisting of the nucleotide sequences set forth in(a)-(b); wherein said nucleotide molecule encodes a P-glycoprotein thatcontrols plant growth or said nucleotide molecule is complementary to anucleotide sequence that encodes said P-glycoprotein.
 5. A transformedplant comprising stably incorporated into its genome a nucleic acidmolecule operably linked to a promoter that drives expression in a plantcell, wherein said nucleic acid molecule comprises a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence havingat least 90% identity to the sequence set forth in SEQ ID NO: 7; (b) anucleotide sequence having at least 90% identity to the sequence setforth in SEQ ID NO: 8; (c) a nucleotide sequence that hybridizes understringent conditions to at least one nucleotide sequence selected fromthe group consisting of the nucleotide sequence set forth in SEQ ID NO:7 and the nucleotide sequence set forth SEQ ID NO: 8, said stringentconditions comprising hybridization at 37° C. in 50% formamide, 1 MNaCl, and 1% SDS and a wash in 0.1×SSC at 60 to 65° C.; and (d) anucleotide sequence that is fully complementary to a nucleotide sequenceselected from the group consisting of the nucleotide sequences set forthin (a)-(c); wherein said nucleotide molecule encodes a P-glycoproteinthat controls plant growth or said nucleotide molecule is complementaryto a nucleotide sequence that encodes said P-glycoprotein.
 6. The plantof claim 5, wherein said promoter is selected from the group consistingof tissue-preferred, constitutive, chemically regulatable, andpathogen-inducible promoters.
 7. The plant of claim 5, wherein saidnucleic acid molecule is operably linked to said promoter in theantisense orientation.
 8. The plant of claim 5, wherein said plant is amonocot.
 9. The plant of claim 8, wherein said monocot is selected fromthe group consisting of maize, wheat, rice, sorghum, rye, millet andbarley.
 10. The plant of claim 5, wherein said plant is a dicot.
 11. Theplant of claim 10, wherein said dicot is selected from the groupconsisting of soybeans, sunflowers, safflowers, alfalfa, Brassica sp.,cotton, peanuts and fruit trees.
 12. Transformed seed of the plant ofclaim
 5. 13. Transformed seed of the plant of claim
 6. 14. Transformedseed of the plant of claim
 7. 15. Transformed seed of the plant of claim8.
 16. Transformed seed of the plant of claim
 9. 17. Transformed seed ofthe plant of claim
 10. 18. Transformed seed of the plant of claim 11.19. A method for modifying the growth of a plant, said method comprisingtransforming a plant with a nucleic acid molecule encoding aP-glycoprotein, said nucleic acid molecule operably linked to a promoterthat drives expression of said nucleic acid molecule in said plant, saidnucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence having at least 90%identity to the sequence set forth in SEQ ID NO: 7; (d) a nucleotidesequence having at least 90% identity to the sequence set forth in SEQID NO: 8; (c) a nucleotide sequence that hybridizes under stringentconditions to at least one nucleotide sequence selected from the groupconsisting of the nucleotide sequence set forth in SEQ ID NO: 7 and thenucleotide sequence set forth SEQ ID NO: 8, said stringent conditionscomprising hybridization at 37° C. in 50% formamide, 1 M NaCl, and 1%SDS and a wash in 0.1×SSC at 60 to 65° C.; and (d) a nucleotide sequencethat is fully complementary to a nucleotide sequence selected from thegroup consisting of the nucleotide sequences set forth in (a)-(c);wherein said nucleotide molecule encodes a P-glycoprotein that controlsplant growth or said nucleotide molecule is complementary to anucleotide sequence that encodes said P-glycoprotein, and wherein thegrowth of said transformed plant is modified.
 20. The method of claim19, wherein said nucleic acid molecule is operably linked to saidpromoter in the antisense orientation.
 21. The method of claim 19,wherein the height of said plant is reduced.
 22. The method of claim 19,wherein the transformed plant has a stable dwarf phenotype.
 23. Themethod of claim 19, wherein said plant is a monocot.
 24. The method ofclaim 23, wherein said monocot is selected from the group consisting ofmaize, wheat, rice, sorghum, rye, millet and barley.
 25. The method ofclaim 22, wherein said transformed plant is a stable dwarf sorghumplant.
 26. The method of claim 25, wherein said stable dwarf sorghumplant is used in commercial sorghum production.
 27. A transformed plantcell comprising stably incorporated into its genome a nucleic acidmolecule operably linked to a promoter that drives expression in a plantcell, wherein said nucleic acid molecule comprises a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence havingat least 90% identity to the sequence set forth in SEQ ID NO: 7; (b) anucleotide sequence having at least 90% identity to the sequence setforth in SEQ ID NO: 8; (c) a nucleotide sequence that hybridizes understringent conditions to at least one nucleotide sequence selected fromthe group consisting of the nucleotide sequence set forth in SEQ ID NO:7 and the nucleotide sequence set forth SEQ ID NO: 8, said stringentconditions comprising hybridization at 37° C. in 50% formamide, 1 MNaCl, and 1% SDS and a wash in 0.1×SSC at 60 to 65° C.; and (d) anucleotide sequence that is fully complementary to a nucleotide sequenceselected from the group consisting of the nucleotide sequences set forthin (a)-(c); wherein said nucleotide molecule encodes a P-glycoproteinthat controls plant growth or said nucleotide molecule is complementaryto a nucleotide sequence that encodes said P-glycoprotein.